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Amyloid fibrils, insoluble β‐sheets structures that arise from protein misfolding, are associated with several neurodegenerative disorders. Many small molecules have been investigated to prevent amyloid fibrils from forming; however, there are currently no therapeutics to combat these diseases. Mass spectrometry (MS) is proving to be effective for studying the high order structure (HOS) of aggregating proteins and for determining structural changes accompanying protein–inhibitor interactions. When combined with native MS (nMS), gas‐phase ion mobility, protein footprinting, and chemical cross‐linking, MS can afford regional and sometimes amino acid spatial resolution of the aggregating protein. The spatial resolution is greater than typical low‐resolution spectroscopic, calorimetric, and the traditional ThT fluorescence methods used in amyloid research today. High‐resolution approaches can struggle when investigating protein aggregation, as the proteins exist as complex oligomeric mixtures of many sizes and several conformations or polymorphs. Thus, MS is positioned to complement both high‐ and low‐resolution approaches to studying amyloid fibril formation and protein–inhibitor interactions. This review covers basics in MS paired with ion mobility, continuous hydrogen‐deuterium exchange (continuous HDX), pulsed hydrogen‐deuterium exchange (pulsed HDX), fast photochemical oxidation of proteins (FPOP) and other irreversible labeling methods, and chemical cross‐linking. We then review the applications of these approaches to studying amyloid‐prone proteins with a focus on amyloid beta and alpha‐synuclein. Another focus is the determination of protein–inhibitor interactions. The expectation is that MS will bring new insights to amyloid formation and thereby play an important role to prevent their formation. [ABSTRACT FROM AUTHOR]

Both trolleys and war leave innocent victims to suffer death and injury. Trolley problems accounting for the injured, and not only the dead, tease out intuitions about liability that enhance our understanding of the obligation to provide compensation and medical care to civilian victims of war. Like many trolley victims, civilians in war may suffer justifiable, excusable, or negligent harms that demand compensation. Chief among these is collateral harm befalling civilians. Collateral harm is endemic to war and comprises permissible but unavoidable death or injury following necessary and proportionate military operations. Although state armies sometimes offer condolence payments for civilian death, injury, and property loss, they deny liability. Instead, they use compensation to enhance counterinsurgency efforts and assuage feelings of agent regret. As part of the medical rules of eligibility, Coalition forces in Iraq and Afghanistan also provided medical care to victims of collateral harm. However, they denied care to similarly sick or injured civilians. While compensation is often justified to cure the harm civilians suffer, the differential use of medical resources is not. Rather, medical care remains subject to the principle of beneficence and medical need. The duty to provide civilian healthcare in war, particularly in wars of humanitarian intervention, is far‐reaching and imposes significant costs that military and medical ethics are yet to recognize. [ABSTRACT FROM AUTHOR]

I This is a personal perspective on our contributions to Fourier-transform ion cyclotron resonance (FT-ICR), including many early ones in a collaboration of Charles Wilkins and Michael Gross starting in the mid-1970s and then continuing for many years in a collaboration of Don Rempel and Michael Gross. The outcomes of our interaction, which arose from a simple question on ion fragmentation were a commitment to collaboration and three publications on potential analytical applications of ICR, one on ion energetics (Gross et al., 1974), and another in I JACS i (Wilkins & Gross, 1971) on the styrene ion-molecule reaction. Wilkins seemed disappointed that nothing novel had turned up, but I pointed out that admitting styrene to the ICR trap led to an interesting ion-molecule reaction where C SB 8 sb H SB 8 sb + C SB 8 sb H SB 8 sb SP +• sp -> C SB 16 sb H SB 16 sb SP +• sp , an observable addition complex that was not seen in the ion-molecule reactions of simpler olefins, a subject I was researching in planning ion-chemistry experiments. Marshall and coworkers (May, Grosshans, & Marshall, 1992) identified another critical mass, an ion's I m i / I z i when its cyclotron radius owing to thermal energy is greater than the dimensions of the trap, and the ion can no longer be contained in the trap even prior to excitation. FT-ICR, Fourier-transform ion cyclotron resonance mass spectrometry; GC/MS, gas chromatography/mass spectrometry. gl The pulsed-valve concept worked well for GC/MS. [Extracted from the article]

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non‐canonical orientations, as compared to other proline‐rich motifs. An affinity tag‐purification mass spectrometry approach identified additional PPxPxY‐containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, and exert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30‐independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen‐deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription. SYNOPSIS: Protein mapping studies identified multiple VP30 interacting host protein possessing PPxPxY motifs, including RBBP6, hnRNP L, hnRNPUL1 and PEG10. RBBP6 and hnRNP L prevent VP30‐NP interaction, impair VP30 dephosphorylation and inhibit viral RNA synthesis. Multiple host proteins possessing PPxPxY motifs can bind the Ebola virus VP30 protein.Proteins with the motif PxPPPPxY bind tightly to VP30, compete with the viral nucleoprotein for binding to VP30 and inhibit Ebola viral RNA synthesis.RBBP6 and hnRNP L modulate Ebola virus transcription by preventing the dephosphorylation of VP30.RBBP6 and hnRNP L act as restriction factors and can cooperatively modulate Ebola virus replication. [ABSTRACT FROM AUTHOR]

A free‐radical footprinting approach is described for integral membrane protein (IMP) that extends, significantly, the "fast photochemical oxidation of proteins" (FPOP) platform. This new approach exploits highly hydrophobic perfluoroisopropyl iodide (PFIPI) together with tip sonication to ensure efficient transport into the micelle interior, allowing laser dissociation and footprinting of the transmembrane domains. In contrast to water soluble footprinters, PFIPI footprints both the hydrophobic intramembrane and the hydrophilic extramembrane domains of the IMP vitamin K epoxide reductase (VKOR). The footprinting is fast, giving high coverage for Tyr (100 %) and Trp. The incorporation of the reagent with sonication does not significantly affect VKOR's enzymatic function, and tyrosine iodination does not compromise protease digestion and the subsequent analysis. The locations for the modifications are largely consistent with the corresponding solvent accessibilities, recommending this approach for future membrane protein footprinting. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) has become a global public health concern because it causes fetal microcephaly and other neurological complications in humans. Currently, there are no approved treatments or vaccines for ZIKV infection. We describe here the epitopes for six monoclonal antibodies (mAbs) that bind to domain III of the envelope protein of ZIKV, some of which have therapeutic potential. We show that by using hydrogen–deuterium exchange mass spectrometry (HDX‐MS), we can identify three spatially distinct epitopes for the six mAbs investigated. The HDX‐MS approach identified epitopes for three mAbs that agreed well with previously reported X‐ray crystallography data. The HDX‐MS determined epitopes for the other three anti‐ZIKV mAbs, for which there were no crystal structures, and the epitopes were confirmed by structure‐guided mutagenesis and biolayer interferometry competition binding assay. Our results have implications for the design of vaccine and antibody therapeutics against ZIKV and demonstrate the use of HDX‐MS as a rapid and valid approach for epitope mapping. [ABSTRACT FROM AUTHOR]

Tetraspanins, including CD53 and CD81, regulate a multitude of cellular processes through organizing an interaction network on cell membranes. Here, we report the crystal structure of CD53 in an open conformation poised for partner interaction. The large extracellular domain (EC2) of CD53 protrudes away from the membrane surface and exposes a variable region, which is identified by hydrogen–deuterium exchange as the common interface for CD53 and CD81 to bind partners. The EC2 orientation in CD53 is supported by an extracellular loop (EC1). At the closed conformation of CD81, however, EC2 disengages from EC1 and rotates toward the membrane, thereby preventing partner interaction. Structural simulation shows that EC1‐EC2 interaction also supports the open conformation of CD81. Disrupting this interaction in CD81 impairs the accurate glycosylation of its CD19 partner, the target for leukemia immunotherapies. Moreover, EC1 mutations in CD53 prevent the chemotaxis of pre‐B cells toward a chemokine that supports B‐cell trafficking and homing within the bone marrow, a major CD53 function identified here. Overall, an open conformation is required for tetraspanin–partner interactions to support myriad cellular processes. Synopsis: Crystal structure of CD53 captures an open conformation for partner interaction and reveals the structural regulation mechanism for tetraspanins to organize interaction networks on cell membranes and support myriad cellular processes. Crystal structure of CD53 captures an open conformation poised for partner interaction. This open conformation is supported by a stable EC1‐EC2 interaction.Hydrogen‐deuterium exchange experiments show that CD53 and CD81 use their C‐D variable region in the EC2 domain to bind partners. This variable region approaches the membrane plane in the closed conformation, and through EC2 rotation in the open conformation, the above‐membrane height of this region is raised to permit partner interaction.Partner specificity of tetraspanins is achieved by the various structures and sequences of the C‐D region, by its above‐membrane height that matches with interacting partners, and by interactions through the transmembrane domains.CD53 assists the migration of pre‐B cells towards the CXCL12 chemokine, whereas disrupting the EC1‐EC2 interaction abolishes this migration. Similar EC1 mutations in CD81 impairs the accurate glycosylation of CD19. [ABSTRACT FROM AUTHOR]

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Structural proteomics refers to large‐scale mapping of protein structures in order to understand the relationship between protein sequence, structure, and function. Chemical labeling, in combination with mass‐spectrometry (MS) analysis, have emerged as powerful tools to enable a broad range of biological applications in structural proteomics. The key to success is a biocompatible reagent that modifies a protein without affecting its high‐order structure. Fluorine, well‐known to exert profound effects on the physical and chemical properties of reagents, should have an impact on structural proteomics. In this Minireview, we describe several fluorine‐containing reagents that can be applied in structural proteomics. We organize their applications around four MS‐based techniques: a) affinity labeling, b) activity‐based protein profiling (ABPP), c) protein footprinting, and d) protein cross‐linking. Our aim is to provide an overview of the research, development, and application of fluorine‐containing reagents in protein structural studies. [ABSTRACT FROM AUTHOR]

Described is a novel, laser-initiated radical trifluoromethylation for protein footprinting and its broad residue coverage. .CF3 reacts with 18 of the 20 common amino acids, including Gly, Ala, Ser, Thr, Asp, and Glu, which are relatively silent with regard to .OH. This new approach to footprinting is a bridge between trifluoromethylation in materials and medicinal chemistry and structural biology and biotechnology. Its application to a membrane protein and to myoglobin show that the approach is sensitive to protein conformational change and solvent accessibility. [ABSTRACT FROM AUTHOR]

Spore photoproduct lyase ( SPL) catalyzes the direct reversal of a thymine dimer 5-thyminyl-5,6-dihydrothymine (i.e. the spore photoproduct ( SP)) to two thymine residues in germinating endospores. Previous studies suggest that SPL from the bacterium Bacillus subtilis ( Bs) harbors an unprecedented radical-transfer pathway starting with cysteine 141 proceeding through tyrosine 99. However, in SPL from the bacterium Clostridium acetobutylicum ( Ca), the cysteine (at position 74) and the tyrosine are located on the opposite sides of a substrate-binding pocket that has to collapse to bring the two residues into proximity, enabling the C→Y radical passage as implied in SPL( Bs). To test this hypothesis, we adopted hydrogen/deuterium exchange mass spectrometry ( HDX- MS) to show that C74( Ca) is located at a highly flexible region. The repair of dinucleotide SP TpT by SPL( Ca) is eight-fold to 10-fold slower than that by SPL( Bs); the process also generates a large portion of the aborted product Tp TSO2−. SPL( Ca) exhibits apparent ( D V) kinetic isotope effects ( KIEs) of ~6 and abnormally large competitive (D V/K) KIEs (~20), both of which are much larger than the KIEs observed for SPL( Bs). All these observations indicate that SPL( Ca) possesses a flexible active site and readily undergoes conformational changes during catalysis. [ABSTRACT FROM AUTHOR]

Background: The development of new innovative treatments to prevent and ameliorate AD requires knowledge of molecular mechanisms that are critical to neuronal health. The triggering receptor expressed on myeloid cells 2 (TREM2) receptor is part of a signaling complex that modulates inflammatory responses, phagocytosis and cell survival in microglia, resident immune cells in the brain that play a critical role in clearing misfolded aggregates. Examples include neurotoxic aggregates consisting largely of amyloid beta (Ab) and apoliproteinE (apoE). Both molecules have emerged as important signaling ligands for TREM2. Although TREM2 signaling in microglia is generally associated with beneficial outcomes, intense or prolonged signaling may produce overactivated microglia leading to neuronal damage. Such events may also contribute to other diseases such as Parkinson's or cancers. Furthermore, rare TREM2 variants, most notably R47H and R62H, have been identified that are associated with a significantly increased risk of developing AD. Given these significant roles, TREM2 has emerged as an important yet challenging therapeutic target for AD. Although a number of ligands for TREM2 with relevance to AD have been identified, little is known regarding the molecular details of how TREM2 engages them. Detailed knowledge of the molecular mechanisms underlying TREM2 signaling in microglia is urgently needed to facilitate the development of specific, potent, safe and efficacious therapies for AD that target the TREM2 signaling pathway. The objective of this study was to identify the critical surfaces on TREM2 that mediate interactions with both apolipoproteinE (apoE) and oligomeric amyloid b1‐42 (oAb42) using rigorous structural and biophysical methods. Method: We used structure‐based mutations and comprehensive biolayer interferometry (BLI) analysis to investigate TREM2 interactions with apoE and oAb42. Result: We found that TREM2 utilizes a distal hydrophobic surface, consisting of the CDR1, CDR2, and CDR3 loops, to engage apoE. Surprisingly, we found that this same surface is utilized to engage oAb42. Conclusion: Our results indicate that the hydrophobic site on TREM2 (consisting of the CDR1, CDR2, and CDR3 loops) is the critical ligand‐engaging surface on TREM2. They also indicate that therapeutics that either directly bind or allosterically alter the hydrophobic site on TREM2 should modulate TREM2 signaling as potential AD treatments. [ABSTRACT FROM AUTHOR]

Native spray has the potential to probe biophysical properties of protein assemblies. Here we report an investigation using both ECD top-down sequencing with an FTICR mass spectrometer and ion mobility (IM) measurements on a Q-TOF to investigate the collisionally induced unfolding of a native-like heterogeneous tetrameric assembly, human hemoglobin (hHb), in the gas phase. To our knowledge, this is the first report combining ECD and ion-mobility data on the same target protein assembly to delineate the effects of collisional activation on both assembly size and the extent and location of fragmentation. Although the collision-induced unfolding of the hemoglobin assembly is clearly seen by both IMMS and ECD, the latter delineates the regions that increasingly unfold as the collision energy is increased. The results are consistent with previous outcomes for homogeneous protein assemblies and reinforce our interpretation that activation opens the structure of the protein assembly from the flexible regions to make available ECD fragmentation, without dissociating the component proteins. [ABSTRACT FROM AUTHOR]

Abstract: Monoclonal antibodies (mAbs) are powerful therapeutics, and their characterization has drawn considerable attention and urgency. Unlike small-molecule drugs (150–600Da) that have rigid structures, mAbs (∼150kDa) are engineered proteins that undergo complicated folding and can exist in a number of low-energy structures, posing a challenge for traditional methods in structural biology. Mass spectrometry (MS)-based biophysical characterization approaches can provide structural information, bringing high sensitivity, fast turnaround, and small sample consumption. This review outlines various MS-based strategies for protein biophysical characterization and then reviews how these strategies provide structural information of mAbs at the protein level (intact or top-down approaches), peptide, and residue level (bottom-up approaches), affording information on higher order structure, aggregation, and the nature of antibody complexes. [Copyright &y& Elsevier]

Abstract: Native mass spectrometry (MS), or as is sometimes called “native electrospray ionization” allows proteins in their native or near-native states in solution to be introduced into the gas phase and interrogated by mass spectrometry. This approach is now a powerful tool to investigate protein complexes. This article reviews the background of native MS of protein complexes and describes its strengths, taking photosynthetic pigment–protein complexes as examples. Native MS can be utilized in combination with other MS-based approaches to obtain complementary information to that provided by tools such as X-ray crystallography and NMR spectroscopy to understand the structure–function relationships of protein complexes. When additional information beyond that provided by native MS is required, other MS-based strategies can be successfully applied to augment the results of native MS. [Copyright &y& Elsevier]

"Medicalized" weapons—those that rely on advances in neuroscience, physiology, and pharmacology—offer the prospect of reducing casualties and protecting civilians. They could be especially useful in modern asymmetric wars in which conventional states are pitted against guerrilla or insurgent forces. But may physicians and other medical workers participate in their development? [ABSTRACT FROM AUTHOR]

Two SDS-resistant endochitinases, designated as ASCHI53 and ASCHI61, were isolated from Aeromonas schubertii in a soil sample from southern Taiwan. MALDI-TOF mass measurement indicates the molecular weights of 53,527 for ASCHI53 and 61,202 for ASCHI61. N-terminal and internal amino acid sequences were obtained, and BLAST analysis of the sequences and MS/MS peptide sequencing showed that they were novel proteins. Degradation of chitin by these two endochitinases gave rise to hexameric chitin oligosaccharide, a compound known to have several potent biomedical functions. ASCHI53 and ASCHI61 retained, respectively, 65% and 75%, of their chitinase activity in the presence of 5% SDS and 100% of their activity in the presence of 10% β-mercaptoethanol. These results demonstrate that they are SDS-resistant endochitinases and probably have a rigid structure. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [ABSTRACT FROM AUTHOR]

During the current round of fighting in the Middle East, Israel has provoked considerable controversy as it turned to targeted killings or assassination to battle militants. While assassination has met with disfavour among traditional observers, commentators have, more recently, sought to justify targeted killings with an appeal to both self-defence and law enforcement. While each paradigm allows the use of lethal force, they are fundamentally incompatible, the former stipulating moral innocence and the latter demanding the presumption of criminal guilt. Putting aside the paradigm of law enforcement which demands due process and forbids extra-judicial execution, the only possible avenue for justifying named killings lies in self-defence. While named killings might be defensible on the grounds that there are no other ways to disable combatants when they fight without uniforms, the costs, including the cost of targeted killing emerging as an acceptable convention in its own right, should be sufficient to view the practice with a good deal of caution. [ABSTRACT FROM AUTHOR]

This report details the biochemical features of natural peptides selected by the H-2Kclass I MHC molecule. In normal cell lines, the length of the naturally processed peptides ranged from 8 to 18 amino acids, although the majority were 9-mers (16% were longer than nine residues). The binding motif for the 9-mer peptides was dominated by the presence of a tyrosine at P2 and an isoleucine/leucine at the P9 position. The P2 residue contributed most towards binding; and the short peptides bound better and formed longer-lived cell surface complexes than the long peptides, which bound poorly and dissociated rapidly. The longer peptides did not exhibit this strictly defined motif. Trimming the long peptides to their shorter forms did not enhance binding and conversely, extending the 9-mer peptides did not decrease binding. The long peptides were present on the cell-surface bound to H-2K (Kd) and were not intermediate products of the class I MHC processing pathway. Finally, in two different TAP-deficient cells the long peptides were the dominant species, which suggested that TAP-independent pathways selected for long peptides by class I MHC molecules. [ABSTRACT FROM AUTHOR]

How should physicians act when faced with corporal punishment, such as amputation, or torture? In most cases, the answer is clear: international law, UN resolutions and universal codes of medical ethics absolutely forbid physicians from countenancing torture and corporal punishment in any form. An acute problem arises, however, in decent societies, but not necessarily liberal states, that are, nonetheless, welcome in the world community. The decent society is often governed, in whole or in part, by religious laws, and while these states abridge various human rights they are peace loving, generally tolerant, and offer their citizens wide avenues for political participation. Under these circumstances the prohibition against corporal punishment and torture weakens, often compelling physicians to participate. This is true in two cases. In Rawls’ hypothetical nation of Kazanistan, Islamic law is the order of the day, and amputations and corporal punishment play an integral part in the execution of traditional Islamic justice. In Israel, torture is sometimes used to elicit the information needed to thwart impending terror attacks. In each case, a physician's participation is essential. In light of the near universal condemnation that accompanies torture and corporal punishment, physicians can only appeal to norms anchored in collective well-being and concern for life that override respect for human dignity in these societies. Western societies have consistently rejected this reasoning, but it is part and parcel of life in the decent society. [ABSTRACT FROM AUTHOR]

Abortion, particularly late-term abortion, and neonaticide, selective non-treatment of newborns, are feasible management strategies for fetuses or newborns diagnosed with severe abnormalities. However, policy varies considerably among developed nations. This article examines abortion and neonatal policy in four nations: Israel, the US, the UK and Denmark. In Israel, late-term abortion is permitted while non-treatment of newborns is prohibited. In the US, on the other hand, late-term abortion is severely restricted, while treatment to newborns may be withdrawn. Policy in the UK and Denmark bridges some of these gaps with liberal abortion and neonatal policy. Disparate policy within and between nations creates practical and ethical difficulties. Practice diverges from policy as many practitioners find it difficult to adhere to official policy. Ethically, it is difficult to entirely justify perinatal policy in these nations. In each nation, there are elements of ethically sound policy, while other aspects cannot be defended. Ethical policy hinges on two underlying normative issues: the question of fetal/newborn status and the morality of killing and letting die. While each issue has been the subject of extensive debate, there are firm ethical norms that should serve as the basis for coherent and consistent perinatal policy. These include 1) a grant of full moral and legal status to the newborn but only partial moral and legal status to the late-term fetus 2) a general prohibition against feticide unless to save the life of the mother or prevent the birth of a fetus facing certain death or severe pain and suffering and 3) a general endorsement of neonaticide subject to a parent’s assessment of the newborn’s interest broadly defined to consider physical harm as well as social, psychological and or financial harm to related third parties. Policies in each of the nations surveyed diverging from these norms should be the subject of public discourse and,... [ABSTRACT FROM AUTHOR]

AbstractThe goals of medical ethics education comprise several dimensions: legal duties to secure informed consent, tell the truth and protect confidentiality; objective competencies that include an understanding of DNR regulations and surrogate decision-making procedures; discursive moral skills such as moral sensitivity, reciprocity and moral development that combine into the capacity for moral dialogue and debate; and finally, behavioural goals that challenge moral education to nurture a more humane, sensitive and communicative physician. Part one of this paper describes each of these goals, together with some of the inherent difficulties affecting implementation. Part two presents survey data from medical ethics instructors (n = 126) who were asked about the importance of each goal and their ability to successfully achieve each aim. While each goal is highly rated, only those goals associated with legal duties and objective competencies are thought to be achieved with any degree of relative success. Goals associated with character transformation, unique to medical ethics education, are among the most difficult to achieve. Additional empirical data corroborate these impressions. Moreover, it is not clear that medical ethics education has much to do with good care. Empirical data are scarce and the conceptual relationship between ethics and care remains highly problematic. Part three offers a number of divergent interpretations of care and concludes that they have little to do with medical ethics. There is no reason to expect that medical competence should be affected by moral competence. Medical ethics therefore might then be viewed as an educational enhancement: unnecessary for healthy doctoring but desirable among a small percentage of the profession who maintain an interest in ethical problems. [ABSTRACT FROM AUTHOR]

Discusses patients' rights and paternalism in Israel. Similarity of Israel's patients' rights with the United States Patient Self-Determination Act; Details on the Israeli Patient Rights Act; Emergence of bioethical norms; Definition of paternalism.